The therapeutic benefits of cell-based treatments have often been regarded as modest, borderline, or nil. As a result, clinical translation has not occurred, and considerable uncertainty and skepticism surround the concept of cell therapy. A major factor limiting the benefits of cell therapy (regardless of cell type) is the poor engraftment of the cells, which disappear rapidly after transplantation; because of this, we propose that administering one dose of cells cannot be considered an adequate test of efficacy. However, almost all preclinical and clinical studies of cell therapy performed heretofore have based their assessment of efficacy on the outcome of one cell administration. Our work in the current PPG has shown that 3 doses of c-kitPOS cardiac progenitor cells (CPCs) are strikingly (~3 times) more effective than 1 dose in improving LV function. Building on this prior work, we will test the idea that the problem of poor engraftment can be overcome by repeated cell administrations. We have recently discovered a new population of cells, cardiac mesenchymal cells (CMCs), that appear to be more suitable for widespread use and to have greater therapeutic potential than CPCs. Consequently, we will focus this proposal on CMCs. The overall goal of Project 1 is to thoroughly evaluate the therapeutic effectiveness and mechanism of action of repeated doses of CMCs. Our central hypothesis is that repeated administrations of CMCs dramatically increase the beneficial effects of cell therapy not only in mice but also in large, preclinical animal models (pigs) and in the presence of major co-morbidities (diabetes) via mechanisms that involve the cumulative effects elicited by repetitive bursts of extracellular vesicle (EV) release, resulting in cumulative, favorable modulation of the inflammatory and fibrotic response to CMC transplantation. The specific Aims are: 1) To establish the most effective protocols for administering repeated doses of CMCs or CMC-derived EVs. We will determine how the effects of repeated doses can be maximized by varying the number of doses and the interval between doses, and the impact of diabetes. 2) To generate data necessary for Phase I clinical trials of repeated CMC or EV therapy in patients with heart failure. Using a preclinical porcine model, we will assess the effects of repeated administrations of CMCs or EVs and identify the optimal doses. 3) To elucidate the mechanism of action of repeated doses of CMCs and EVs, including their effect on the inflammatory and fibroblast response to infarction. Throughout these studies, the effects of EVs will be compared with those of CMCs to determine whether cell-free EV therapy recapitulates the benefits of CMC therapy. This Project will test two potentially disruptive ideas: i) the concept that the full effects of cell or EV therapy require repeated doses would constitute a major paradigm shift, with potentially vast implications for the entire field of reparative medicine; ii) the concept that the effects of cell therapy can be reproduced with cell-free administration of EVs would revolutionize the field. These studies will lay the groundwork necessary for first-in-human trials of repeated CMC/EV administration.